1. Field of the Invention
This invention is in the field of chemical and biological assay and is useful for measurement of variables which are addressable through sensitive measurements of the index of refraction.
2. Brief Description of the Prior Art
Evanescent wave spectroscopy is not new. It is a variant of internal reflection spectroscopy and many configurations are thoroughly described in a text by N. J. Harrick. The concept works generally through the illumination of an optical interface which is in contact with a sample material; interaction with the sample occurs through evanescent wave sampling. Some configurations operate near the angle of total internal reflection and the sample index is tested either by measurement of the critical angle or by measurement of light loss as the solution index changes. Additional methods are provided through measurement of evanescent wave adsorption. In bulk optical systems, the light intensity is low and suitable sensitivity is obtained by multiple exposures to the sample interface, usually by geometric constructions which provide multiple reflections.
Some multimode fiber optic variants to the theme are described in Harrick""s book. They operate on the same principal as the bulk optic devices with extended exposure to the fiber/sample solution interface provided by the multibounce propagation method in the multimode fiber. Sensing is via intensity variation at the output.
Three construction variants, based on multimode optical fibers, have been proposed in the literature as candidates for immunoassay. In the first, the fiber cladding is stripped to expose the core, and antigens, labeled with fluorophores, are attached to the fiber surface. The fluorophores are excited by the evanescent field and can be detected through reduction in the light level of by collection of the fluorescence. A second type of fiber optic immunoassay sensor uses a coating deposited on the fiber tip that can be illuminated by an optical pulse, which in turn induces fluorescence which is reflected back up the fiber and detected. A third type of fiber optic sensor for immunoassay involves a stripped fiber core that has antibodies and antigens attached to the core/solution interface. This sensor is used as one leg of a fiber optic Mach-Zender interferometer. The binding of molecules to the surface during attachment of either antibody of antigen suffices to locally change the index of refraction at the core/solution interface. This changes the phase velocity of the light on one leg of the device and interference fringes are observed at its output.
Still other configurations of immunosensors have been described such as surface plasmon resonance immunosensors, and grating couplers, used as integrated optical chemical sensors. These and others are discussed in the book edited by Wolfbeis. Velander and Murphy at Virginia Tech have proposed a fiber optic technique for an immunoassay that uses a grating superimposed on the fiber to scatter light into the cladding where it can sample the cladding/air interface. An affinity aerogel coating is used to collect and concentrate target antigens which are measured through the absorption spectrum of the returned light. Still more methods based on evanescent field absorption in optical waveguides are described by G. Stewart.
Conventional evanescent wave spectrometry has been thoroughly researched and is well known in the literature. The techniques involved are also used in fiber optic sensing. The processes usually rely on absorption processes in regions of waveguides where the evanescent field penetrates the guide. The guides are arranged so that as many reflections as possible illuminate the sample interface. Even so, the places where ray optics allows interaction between the optical species and the sample arc comparatively few and the illumination is weak compared to the single mode fiber optic coupler approach.
In large multimode optical fibers, a relatively large number, possibly hundreds, of spatial modes are supported. The modes can and do interfere with each other leading to extensive noise generation at the point of detection (i.e. speckle). The detector can""t distinguish between intensity variations due to the sample and intensity variations that occur due to random inference between the propagating optical modes.
In contrast, single mode fibers only support one propagating mode. Therefore random interference is impossible and no modal redistribution occurs due to environmental factors. Another significant advantage of single mode fibers is that more than 90% of the optical energy can be forced into the evanescent field and that field surrounds the entire space immediately surrounding the core.
Interferometric approaches are usually the most sensitive available, however they require exceptional mechanical stability. In the fiber optic case, the interferometric technique suffers because the optical signal rotates due to birefringence in the bent fiber and path stability becomes a phenomenal problem. The single mode fiber optic coupler sensor is also an interferometric device, of sorts. However, the two legs of the interferometer (the two propagating supermodes) are both contained within the device itself. Because of this, the device is self-referencing and the noise associated with path instability is completely avoided.
The technique used by Veander and Murphy requires a spectrophotometer to read. Even in miniature configuration, this is hardly a xe2x80x9cpoint of carexe2x80x9d device useful in field environments.
In a prior patent, Gerdt and Herr correctly described the significant benefits of the single mode fiber optic coupler sensor relative to current and prior art. The disclosure of the prior patent. U.S. Pat. No. 5,494,798, Gerdt and Herr, Feb. 27, 1996, is incorporated by reference herein, as though recited in full. To summarize, the benefits include: 1) the high illumination levels exposed to the sample interface and the high sensitivity which result; 2) the single mode field exposed to the sample and the low noise which results with the elimination of other interfering modal noise components in the measured signal; 3) a measurement which uses only the variation in propagation constant to sense the measurement and is inherently separable from the multitude of intensity noise sources which encumber the measurement; 4) a differential signal output which allows normalization of the measurement and isolation from other noise sources.
In their patent, Gerdt and Herr proposed a method for immunoassay using the single mode fiber optic coupler sensor in conjunction with a surface coating of specific monoclonal or polyclonal antibodies to detect small concentrations of target antigens.
There are three significant weaknesses in the method proposed which limit the ultimate sensitivity of the device and its use for quantitative measurement. The first occurs because the optical source produces noise components in both intensity and frequency. In their proposed method, the intensity noise is removed by conventional difference/sum signal processing of the optical output. Although the intensity noise components are removed by the method, the frequency noise components are enhanced along with the real signal. No method was provided to address this noise component that becomes significant at higher values of solution index of refraction. The second limitation occurs because the measured power splitting ratio is transcendental. Single measurements of the splitting ratio are not unique and no method is provided to quantitatively assess the solution index of refraction. The third limitation occurs because the gain and thus the noise figure in the initial detection stage is limited by the need to accept the full optical signal without clipping at large values of the coupling ratio which occurs periodically as the solution index is changed. As a result, small signal changes must be measured on a very large background. The signal, thus measured, is very small and system sensitivity suffers as the result. The proposed system, as illustrated in FIG. 1, includes a reference channel that addresses these shortcomings of the proposed method of the prior art.
The invention relates broadly to a fiber optic sensor device, advantageously, a refractive index measurement device. A light emitting member transmits light, preferably coherent light, to a light splitter. The light splitter splits the light into a first portion that is transmitted to a first fiber optic coupler having an input optical fiber member and an optical fiber member having a waist region. The coupler preferably is formed from a plurality of single mode fiber optic fibers. A first output optical fiber member emerges from the first fiber optic coupler waist region. The light splitter is positioned to insert light into the first fiber optic coupler input optical fiber member.
A reference fiber optic coupler includes an input optical fiber member and an optical fiber member having a waist region. The reference fiber optic coupler is enclosed in a potting medium stable reference of constant refractive index. A reference output optical fiber member emerges from the reference fiber optic coupler waist region. The first fiber optic coupler and the reference fiber optic coupler are single mode couplers.
The light splitter member inserts a second portion of the split light into the reference fiber optic coupler input optical fiber member.
A light measuring member is optically coupled to receive light emitted from the first output optical fiber member and the reference output optical fiber member. The light measuring member can be a light meter for measuring the magnitude of light.
A sum difference device processes the measured light emitted from the first output optical fiber member and from the reference output optical fiber member whereby the value derived said measured light emitted from said first output optical fiber member is independent of variations in light from said light emitting member.
The device of the present invention can be used for the immunoassay measurement of a target component. Light output from two or more of the sample coupler optic fibers are measured before and after surrounding the coupler with the target component. Light is measured from the reference coupler to establish a light reference value. The presence or concentration of the target component is determined by measuring changes in the output of light from the two or more optic fibers of the sample coupler due to the specific binding of the target component to the first immunoassay component as compared to the light reference value. The step of measuring changes in the output light preferably comprises a plurality of measurements made over a predetermine time interval and determining the rate of change of the light output. The step of comparing preferably includes comparing the rate of change of the light output as an indication of the concentration of the target material in the fluid.
The present invention advantageously includes an immunoassay method for determining a target analyte in a fluid sample. The method does not use labeled reagents and is based upon measuring changes in refractive index in an evanescent field surrounding a fiber optic coupler relative to light changes in a reference fiber optic coupler surrounded by a medium of stable refractive index. The method includes immobilizing an antibody or antigen capable of specifically binding to the target analyte on the fiber optic coupler and illuminating at least one of the plurality of optical fibers to provide the surrounding evanescent field with a first portion of light from a light splitter.
A first refractive index of the surrounding evanescent field is obtained by measuring a first ratio of light output by the fiber optic coupler. The fluid sample is brought into contact with the target fiber optic-coupler such that any target analyte in the fluid sample specifically binds to the immobilized antibody or antigen. A second refractive index of the surrounding field is obtained by measuring a second ratio of light output by the target fiber optic coupler.
The reference coupler is illuminated with a second portion of light from the light splitter to determine a illumination reference level by measuring light output from said reference coupler